Mutational Analysis of a Type II Topoisomerase Cleavage Site: Distinct Requirements for Enzyme and Inhibitors

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 1993 May 1

PMID 8387918

Citations 14

Authors

C H Freudenreich

K N Kreuzer

Affiliations

Soon will be listed here.

Abstract

We have analyzed the DNA sequence requirements for cleavage of a 30 bp oligonucleotide that contains a strong bacteriophage T4 type II topoisomerase site. A novel method was used to generate substrates with each of the four nucleotides at 10 positions surrounding the cleavage site, and mutant substrates were also prepared for the four internal positions of the staggered cleavage site. The substrates were tested for cleavage in the presence of several inhibitors that induce enzyme-mediated cleavage: four antitumor agents of different classes (an aminoacridine, a substituted anthraquinone, an ellipticine derivative and an epipodophyllotoxin) and one antibacterial quinolone. At eight nucleotide positions flanking the cleavage site, the same preferred bases were found regardless of which inhibitor was present. These preferred bases show dyad symmetry with respect to the cleavage site, indicating that both protomers of the topoisomerase homodimer interact with DNA in an analogous manner. In addition, we found that the preferred bases on the 5' side of each cleaved phosphodiester bond are highly specific to the inhibitor used in the cleavage reaction. These results strongly suggest that the inhibitors interact directly with the DNA bases at the cleavage site, placing the inhibitor binding site precisely at the site of DNA cleavage.

Citing Articles

Functional determinants of gate-DNA selection and cleavage by bacterial type II topoisomerases.

Arnoldi E, Pan X, Fisher L Nucleic Acids Res. 2013; 41(20):9411-23.

PMID: 23939623 PMC: 3814380. DOI: 10.1093/nar/gkt696.

Isolation and characterization of mAMSA-hypersensitive mutants. Cytotoxicity of Top2 covalent complexes containing DNA single strand breaks.

Rogojina A, Nitiss J J Biol Chem. 2008; 283(43):29239-50.

PMID: 18723844 PMC: 2570872. DOI: 10.1074/jbc.M804058200.

Position-specific trapping of topoisomerase II by benzo[a]pyrene diol epoxide adducts: implications for interactions with intercalating anticancer agents.

Khan Q, Kohlhagen G, Marshall R, Austin C, Kalena G, Kroth H Proc Natl Acad Sci U S A. 2003; 100(21):12498-503.

PMID: 14523238 PMC: 218786. DOI: 10.1073/pnas.2032456100.

Bacteriophage T4 genome.

Miller E, Kutter E, Mosig G, Arisaka F, Kunisawa T, Ruger W Microbiol Mol Biol Rev. 2003; 67(1):86-156, table of contents.

PMID: 12626685 PMC: 150520. DOI: 10.1128/MMBR.67.1.86-156.2003.

Repair of topoisomerase-mediated DNA damage in bacteriophage T4.

Stohr B, Kreuzer K Genetics. 2001; 158(1):19-28.

PMID: 11333215 PMC: 1461630. DOI: 10.1093/genetics/158.1.19.

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